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A cluster of thin tubular structures mediates transformation of the endoplasmic reticulum to autophagic isolation membrane.

ABSTRACT: Recent findings have suggested that the autophagic isolation membrane (IM) might originate from a domain of the endoplasmic reticulum (ER) called the omegasome. However, the morphological relationships between ER, omegasome, and IM remain unclear. In the present study, we found that hybrid structures composed of a double FYVE domain-containing protein 1 (DFCP1)-positive omegasome and the IM accumulated in Atg3-deficient mouse embryonic fibroblasts (MEFs). Moreover, correlative light and electron microscopy and immunoelectron microscopy revealed that green fluorescent protein (GFP)-tagged DFCP1 was localized on tubular or vesicular elements adjacent to the IM rims. Through detailed morphological analyses, including optimization of a fixation method and electron tomography, we observed a cluster of thin tubular structures between the IM edges and ER, part of which were continuous with IM and/or ER. The formation of these thin tubular clusters was observed in several cell lines and MEFs deficient for Atg5, Atg7, or Atg16L1 but not in FIP200-deficient cells, suggesting that they were relevant to the earlier events in autophagosome formation. Taken together, our findings indicate that these tubular profiles represent a part of the omegasome that links the ER with the IM.

SUBMITTER: Uemura T

PROVIDER: S-EPMC3993601 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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A cluster of thin tubular structures mediates transformation of the endoplasmic reticulum to autophagic isolation membrane.

Uemura Takefumi T Yamamoto Masaya M Kametaka Ai A Sou Yu-shin YS Yabashi Atsuko A Yamada Akane A Annoh Hiromichi H Kametaka Satoshi S Komatsu Masaaki M Waguri Satoshi S

Molecular and cellular biology 20140303 9

Recent findings have suggested that the autophagic isolation membrane (IM) might originate from a domain of the endoplasmic reticulum (ER) called the omegasome. However, the morphological relationships between ER, omegasome, and IM remain unclear. In the present study, we found that hybrid structures composed of a double FYVE domain-containing protein 1 (DFCP1)-positive omegasome and the IM accumulated in Atg3-deficient mouse embryonic fibroblasts (MEFs). Moreover, correlative light and electron ...[more]

PMID: 24591649

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Project description:ObjectiveThis study aimed to explore the molecular mechanism of cytoplasmic vacuolation caused by BK polyomavirus (BKPyV) and thus search for potential target for drug repurposing.MethodsMorphological features of BK polyomavirus-associated nephropathy (BKPyVAN) were studied under light and electron microscopes. Microarray datasets GSE75693, GSE47199, and GSE72925 were integrated by ComBat, and differentially expressed genes (DEGs) were analyzed using limma. Furthermore, the endoplasmic reticulum (ER)-related genes obtained from GenCLiP 2.0 were intersected with DEGs. GO and KEGG enrichment pathways were performed with intersection genes by R package clusterProfiler. The single-cell RNA sequencing (scRNA-seq) from a BKPyVAN recipient was analyzed with a dataset (GSE140989) downloaded from Gene Expression Omnibus (GEO) as control for gene set variation analysis (GSVA). Immunohistochemistry and electron microscopy of kidney sections from drug-induced ERS mouse models were performed to explore the association of ERS and renal tubular vacuolation. Protein-protein interaction (PPI) network of the intersection genes was constructed to identify hub target. AutoDock was used to screen Food and Drug Administration (FDA)-approved drugs that potentially targeted hub gene.ResultsLight and electron microscopes exhibited obvious intranuclear inclusions, vacuoles, and virus particles in BKPyV-infected renal tubular cells. Transcriptome analysis revealed 629 DEGs between samples of BKPyVAN and stable transplanted kidneys, of which 16 were ER-associated genes. GO analysis with the intersection genes illustrated that ERS-related pathways were significantly involved, and KEGG analysis showed a prominent enrichment of MAPK, Toll-like receptor, and chemokine signaling pathways. GSVA analysis of the proximal tubule revealed similar pathways enrichment. An electron microscope image of the kidney from ERS mouse models showed an obvious renal tubular vacuolation with prominent activation of ERS markers verified by immunohistochemistry. Furthermore, DDIT3 was identified as the hub gene based on PPI analysis, and ZINCOOOOO1531009 (Risedronate) was indicated to be a potential drug for DDIT3.ConclusionERS was involved in renal tubular cytoplasmic vacuolation in BKPyVAN recipients. Risedronate was screened as a potential drug for BKPyVAN by targeting DDIT3.

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A cluster of thin tubular structures mediates transformation of the endoplasmic reticulum to autophagic isolation membrane.

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A cluster of thin tubular structures mediates transformation of the endoplasmic reticulum to autophagic isolation membrane.

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